Use of leptin for treating human lipoatrophy and method of determining predisposition to said treatment

ABSTRACT

Leptin, leptin analogs, and leptin derivatives are used to treat patients with lipoatrophy. Leptin is effective against conditions of lipoatrophy for both genetic and acquired forms of the disease. A therapeutically effective amount of leptin can be administered in a variety of ways, including subcutaneously and using gene therapy methods. Methods of the present invention contemplate administration of leptin, leptin analogs, and leptin derivatives to patients having a leptin level of approximately 4 ng/ml or less before treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present continuation application claims priority to U.S. Utilityapplication Ser. No. 10/279,129, filed Oct. 22, 2002, which claimspriority to U.S. Provisional Application Ser. No. 60/336,394 (DePaoli etal.), filed Oct. 22, 2001, the disclosures of which are incorporated byreference in their entirety herein.

GOVERNMENT INTEREST

The present invention was supported, in part, by funding from the NIH.The government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic use of leptin,leptin analogs, and leptin derivatives for the treatment of humanlipoatrophy.

BACKGROUND OF THE INVENTION

All citations herein are incorporated in their entirety by reference.Full citations of the references can be found at the end of the detaileddescription.

The lipoatrophy (also known as lipodystrophy) syndromes are aheterogeneous group of syndromes characterized by a paucity of adiposeor fat tissue. Metabolic abnormalities may also be associated with thiscondition. These metabolic abnormalities include hypertriglyceridemiaand severe insulin resistance usually accompanied by diabetes mellitus(Reitmann et al., 2000). Lipoatrophy in humans may be geneticallyinherited or acquired. There is more than one genetic form oflipoatrophy. For example, mutations in the gene encoding lamin A/C(LMNA) has been shown to be associated with the Dunnigan-type familialpartial lipodystrophy (FPLD) (Cao et al., 2000). Individuals withDunnigan's FPLD are born with a normal fat distribution, but at puberty,they develop progressive subcutaneous extremity and truncal fat loss,with sparing of visceral and head and neck adipose tissue. A differentchromosomal location (9q34) has also been linked to a disease gene forcongenital generalized lipodystrophy (Garg et al., 1999). Congenitalgeneralized lipodystrophy is a recessive disorder characterized by anear complete absence of adipose tissue from birth, insulin resistance,hypertriglyceridemia and acanthosis nigricans.

Some forms of lipoatrophy in humans are acquired. For example, manypatients infected with human immunodeficiency virus (HIV) and treatedwith highly active antiretroviral therapy (HAART) develop a partiallipodystrophy, characterized by loss of subcutaneous fat from the face,extremities and trunk, with increased visceral fat and a ‘buffalo hump’similar to that seen in Cushing's syndrome. These patients may alsodevelop metabolic disorders such as insulin resistance andhypertriglyceridemia. Acquired forms of lipoatrophy may also beassociated with juvenile dermamyositis and other autoimmune diseases.

Investigations in animal models have demonstrated that these metabolicabnormalities may be associated with fat loss (Gavrilova et al., 2000).But insulin resistance and hypertriglyceridemia that characterizelipoatrophy have been extremely refractory to treatment, even though avariety of approaches have been tried (Garg, 2000). One of theseapproaches includes treatment with thiazolidinediones, which we PPARγ(peroxisome proliferator activated receptor γ) agonists. Whilethiazolidinediones are appealing because they promote both adipocytedifferentiation and insulin sensitivity, patients receivingthiazolidinediones are usually managed with combination therapy,including high dose insulin, oral hypoglycemic agents (e.g. metforminand thiazolidinediones), and lipid-lowering drugs, (e.g., fibrates andstatins). Despite these therapies, patients with generalized lipoatrophycontinue to manifest severe hypertriglyceridemia (which causes recurrentattacks of acute pancreatitis), severe hyperglycemia (which poses riskof diabetic retinopathy and nephropathy), and non-alcoholicsteatohepatitis (which can result in cirrhosis) (Arioglu et al., 2000).In fact, one member of the thiazolidinediones, troglitazone, was removedfrom the US market because of its rare but severe hepatotoxicity,leaving two thiazolidinediones (rosiglitazone and pioglitazone)available (Reitmann, et al.). Thus, there exists a need for analternative treatment to lipoatrophy.

A variety of genetically engineered animal models for lipoatrophy havebeen developed and tested. These models, however, provide conflictingresults as to the sensitivity of these animals to treatment with leptin.For example, in one transgenic mouse model, which expresses a truncatednuclear version of SREBP-1 c and mimics the features of congenitalgeneralized lipodystrophy having insulin resistance and markedly lowadipose tissue, continuous systemic infusion of leptin overcame theresistance of the mice to insulin (Shimomura et al., 1999). On the otherhand, a different transgenic mouse, which expresses the A-ZIP/F-I geneand characterized by lack of fat tissue, severe resistance to insulin,diabetes, and greatly reduced serum leptin levels, failed to respond toleptin at similar doses and were minimally effective at higher doses(Gavrilova et al., 2000). Any efficacy with leptin also diminished withage of the animal (Id.). Furthermore, although insulin resistance wasovercome with leptin in the SREBP-1c transgenic mice, reversal oflipoatrophy was not observed (Shimomura et al.).

Current use of leptin in human therapy has mainly been focused onreducing obesity and its associated metabolic dysfunction (Heymsfield etal. 1999). Patients with absence of leptin due to mutations in theleptin gene are morbidly obese from infancy and have a number ofhormonal abnormalities including insulin resistance and hypogonadotropichypogonadism (Montague et al., 1997). Physiological replacement withrecombinant leptin for one year in one of these patients causedsignificant weight reduction and improvement in the hormonalabnormalities (Farooqi et al., 1999; PCT App. No.: WO 00/20872). Theseprevious studies have not addressed the use of leptin in the context ofhuman lipoatrophy.

SUMMARY OF THE INVENTION

The present invention provides for the use of leptin in treating humanswith lipoatrophy and its associated metabolic abnormalities, andprovides a method of determining a predisposition to leptin treatment.In one embodiment, human leptin is used in hormone replacement therapyin lipoatrophic patients having reduced serum concentration of leptin.Preferably, recombinant human leptin or leptin analog or derivative isused. Leptin proteins may be administered subcutaneously orsystemically, or through any other routes including methods in genetherapy.

In assessing the predisposition of lipoatrophic patient to treatmentwith leptin, serum concentration of leptin may be determined.Preferably, patients with serum leptin concentration of less than 4ng/ml, and more preferably, less than 2 ng/ml, and most preferred, lessthan 0.5 ng/ml, are subjected to leptin treatment. It is also preferredthat treatment with leptin be given to female patients with <4 ng/ml ofserum leptin concentration and to male patients with <3 ng/ml of serumleptin concentration. More preferably, leptin is given to male patientswith <2 ng/ml of serum leptin concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the clinical course of patient NIH-1 with 4 months ofleptin therapy. Historical data before leptin therapy (started at Day 0)is presented to demonstrate the severity of metabolic findings.Important milestones of therapy and the improvement in metabolicparameters are shown. FIG. 1B depicts a TI-weighted axial magneticresonance imaging of patient NIH-1 at the level of L4 at baseline and at4-months of leptin therapy. Note the decrease in liver size, and theconsequent changes in position of the kidneys and midline structures.

FIG. 2 shows that leptin decreases HbA_(1c) in the diabetic patients(n=8). Data are presented as mean changes and error bars indicate 95%Confidence Interval. The baseline and 4-month value±SEM (standard errorof the mean) are also shown. *p<0.001.

FIG. 3 shows that leptin improves the glucose curve during both insulintolerance and oral glucose tolerance (n=9). Panel A: Plasma glucose inresponse to 0.2 U.kg IV insulin before (closed circles and solid line)and 4-months after (open circles and dotted line) leptin therapy. Errorbars indicate SEM *p<0.02. Panel B: Plasma glucose in response to75-gram oral glucose before (closed circles and solid line) and 4-monthsafter (open circles and dotted line) leptin therapy. Error bars indicateSEM. *p<0.01.

FIG. 4 shows that leptin decreases triglycerides. Data are presented asmean change from baseline and error bars represent 95% ConfidenceInterval. The mean baseline and 4-month values with observed ranges arealso shown. Note that data are skewed and do not follow a normaldistribution. *p<0.001.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The adipocyte hormone leptin plays a central role in energy homeostasis.It was first discovered in the obese mouse as the missing serum factorthat decreased food intake and body weight upon replacement (Zhang etal., 1994; Pelleymounter et al., 1995). Because of these initialobservations, much of the earlier therapeutic attempt using this hormonehas been in the treatment of obesity. Serum leptin concentrations in themajority of humans with obesity are high, and a state of leptinresistance is thought to exist (Mantzoros et al., 2000). Thus far, theeffect of recombinant human leptin has been limited in causing weightloss in obese individuals except in the state of congenital leptindeficiency (Heymsfield et al., 1999; Farooqi et al., 1999).

The present invention provides for the feasibility of using leptin forthe treatment of lipoatrophy and its associated metabolic abnormalitiesin humans such as hyperglycemia, dyslipidemia, hyperlipidemia,hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascularrestenosis, and insulin resistance. Results from studies in HIV patientshave shown that decrease in serum concentrations of leptin is closelyassociated with the onset of acquired lipoatrophy. Furthermore, leptinreplacement in lipoatrophic patients dramatically improves glucose andtriglyceride metabolism even after all other potential therapies havebeen extinguished. In all these leptin replacement therapy cases, thebaseline serum concentration of leptin was less than 4 ng/ml.

In one severe case of acquired lipoatrophy, the patient (having serumleptin concentration of <0.5 ng/ml) suffered from severehypertriglyceridemia, diabetes, painful eruptive cutaneous xanthomata,and massive hepatomegaly. Leptin treatment over four months dramaticallyimproved the patient's hypertriglyceridemia and hyperglycemia thatallowed for discontinuation of plasmapheresis and other diabetesmedications. The improvements were also accompanied by disappearance ofthe cutaneous xanthomata and the patient's liver volume decreased by40%. Thus, these data show that leptin replacement therapy mayeffectively be used to treat acquired or congenital lipoatrophy and itsassociate metabolic abnormalities in human.

Furthermore, based on these data, it may be extrapolated that patientswith less than 4 ng/ml serum concentration for leptin may be a preferredgroup of patients for replacement therapy with leptin. Leptin levels maybe measured using a body fluid, most preferably blood or some portionthereof. Here, serums from individuals were used. Other body fluids mayalso contain measurable leptin, such as whole blood, cerebral spinalfluid, plasma, and possibly urine. The present measurements of 4 ng ofleptin/ml of serum may be correlated to corresponding levels in otherbody fluids. For example, if whole blood is used, the leptinconcentration will be diluted to account for the diluting effect ofusing unfractionated blood.

One skilled in the art will be able to ascertain effective dosages byadministering leptin, leptin analog or leptin derivative and observingthe desired therapeutic effect. The goal of replacement therapy is toachieve near physiological concentrations of leptin in the plasma. It isestimated that the physiological replacement dose of leptin is about0.02 mg per kilogram of body weight per day for males of all ages, about0.03 mg per kilogram per day for females under 18 years and about 0.04mg per kilogram per day for adult females. When attempting to achievenear physiological concentrations of leptin, one may, for example, treata patient with 50 percent of the estimated replacement dose for thefirst month of treatment, 100 percent of the replacement dose for thesecond month of treatment, 200 percent of the replacement dose for thethird month of treatment, etc. During the course of leptin replacementtherapy, one can measure certain biochemical markers to monitortherapeutic effect of the leptin treatment. Glycosylated hemoglobin(HbA_(1c)) levels and triglyceride (fasting) levels are among thepreferred markers to measure therapeutic effect to monitor the efficacyof leptin treatment.

Alternatively, serum leptin levels can be measured using commerciallyavailable immunoassays, as further disclosed in the Examples below. Ingeneral, a diagnostic assay for measuring the amount of leptin in theblood (or plasma or serum) may first be used to determine endogenouslevels of protein. Such diagnostic tools may be in the form of anantibody assay, such as an antibody sandwich assay. The amount ofendogenous leptin is quantified initially, and a baseline is determined.The therapeutic dosages are determined as the quantification ofendogenous and exogenous leptin protein (that is, leptin, leptin analogor leptin derivative found within the body, either self-produced oradministered). Monitoring the leptin levels of a patient is continuedover the course of therapy.

The present invention also provides methods of using pharmaceuticalcompositions of leptin, leptin analog or leptin derivative. Suchpharmaceutical compositions may be for administration for injection, orfor oral, pulmonary, nasal, transdermal or other forms ofadministration. Preferred methods of administering the leptin proteinsinclude subcutaneously, systemically and by gene therapy methods.

In general, pharmaceutical compositions of the present inventioncomprise effective amounts of leptin, leptin analog or leptin derivativetogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hylauronic acid may also be used, and this may havethe effect of promoting sustained duration in the circulation. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present proteins andderivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which areherein incorporated by reference. The compositions may be prepared inliquid form, or may be in dried powder, such as lyophilized form.Implantable sustained release formulations are also contemplated, as aretransdermal formulations.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives that potentially enhance uptake of the leptin, leptin analogor leptin derivative protein are for instance the fatty acids oleicacid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The leptin, leptinanalog or leptin derivative protein could be incorporated into an inertmatrix which permits release by either diffusion or leaching mechanismse.g., gums. Slowly degenerating matrices may also be incorporated intothe formulation, e.g., alginates, polysaccharides. Another form of acontrolled release of this therapeutic is by a method based on the Orostherapeutic system (Alza Corp.), i.e., the leptin, leptin analog orleptin derivative protein is enclosed in a semi-permeable membrane,which allows water to enter and push the protein out through a singlesmall opening due to osmotic effects. Some enteric coatings also have adelayed release effect.

Further, improved kits for determining the predisposition of a humanpatient with lipoatrophy to respond to treatment with leptin, leptinanalog or leptin derivative are contemplated by the present invention.In one aspect, an improved kit may provide means for determining whetherthe leptin level of the patient prior to said leptin treatment is lessthan or equal to approximately 4 ng/ml. In a related aspect, an improvedkit may consider the gender of a patient when determining a leptin levelin the patient prior to said leptin treatment. Then, the kit may providemeans for determining whether the leptin level of the patient prior tosaid leptin treatment is less than or equal to approximately 2 ng/ml ifthe patient is male, or less than or equal to approximately 4 ng/ml ifthe patient is female. Preferably, the kit comprises instructions foruse. The kit may also comprise reagents, tubes, packaging, and/or otherreaction components.

The following descriptions are provided only as examples and should notbe understood to be limiting on the claims. Based on the description, aperson skilled in the art may make modification and changes to thepreferred embodiments, which do not depart from the scope of theinvention.

Example I

The following example shows that the development of HIV-associatedlipoatrophy syndrome (HIV-LS) may be influenced by reduced leptin in theserum, which contributes to the accumulation, loss or redistribution ofbody fat.

In particular, a study was conducted for the purpose of determiningwhether the lipoatrophy phenotype in HIV-LS is associated with changesin serum leptin following initiation of highly active antiretroviraltherapy (HAART). This study included one hundred forty six (146) HIVpositive men whose serum leptin concentrations were compared before andafter HAART. By physical examination, the men were assessed andstratified into the two major phenotypes: lipoatrophy alone andlipoatrophy with central fat gain (“mixed” HIV-LS).

Out of the 146 men, forty-two (42/146) men were found to have moderateor severe lipoatrophy or lipohypertrophy in more than one body areafollowing HAART. Twenty-seven of the 146 (27/146) had lipoatrophy aloneand fifteen (15/146) had “mixed” changes after HAART. Thirty-nine out ofthe 146 (39/146) did not have body habitus changes and these patientsserved as controls. Generally, the men with HIV-LS were older and hadlonger use of protease inhibitors. They also had lower baseline CD4counts and had lost an average of 4 kg body weight from baseline.

Before HAART, median baseline leptin levels for both the lipoatrophy and“mixed” groups were 3.6 ng/ml and median leptin level for the controlwas 4.1 ng/ml. In those who developed lipoatrophy alone after HAART,serum leptin concentration decreased significantly from 3.6 to 2.8 ng/ml(Wilcoxon p=0.006). On the other hand, serum leptin levels remainedstable in both the “mixed” HIV-LS group (4.0 ng/ml) [p=NS] and in the 39HIV positive controls who did not develop HIV-LS (3.7 ng/ml) [p=NS].

These data suggest that a reduced leptin level following the highlyactive antiretroviral therapy in HIV positive patients may contribute tothe development of lipoatrophy syndrome.

Example II

To determine the efficacy of using leptin to treat lipoatrophy inhumans, leptin replacement therapy was also conducted in nine femalepatients who have been diagnosed with various forms of lipoatrophy. Thepatients of this study were referred by multiple physicians in theUnited States and in Europe. To be eligible, the patients were requiredto have low levels (defined as serum leptin concentration of <3.0 ng/mlin males and <4.0 ng/ml in females) in association with lipodystrophy,and at least one of the following metabolic abnormalities: (1) Presenceof diabetes mellitus by American Diabetes Association criteria (SeePeters et al., 1998); (2) fasting serum triglyceride concentrations >200mg/dL; and/or (3) fasting serum insulin concentrations >30 μU/ml. Thediagnosis of lipodystrophy was based on clinical grounds well known toone ordinary skilled in the art.

Table 1 summarizes the baseline clinical characteristics of the patientstreated in the study. TABLE 1 Characteristics of Patients FastingLipid-Lowering Insulin¹ Leptin² RMR³ Total Fat⁴ Patient Age/Sex/TypeTherapy (μU/mL) (ng/mL) (kcal/day) (%) NIH-1 17/F Fenoflbrate 31.2 <0.52010 7 Acquired Atorvastatin Generalized Orlistat, Weekly PlasmapharesisNIH-2 17/F None 334 1.0 2030 17 Congenital Generalized NIH-3 27/F None19 0.7 1570 18 Acquired Generalized NIH-4 17/F None 211 1.1 2480 17Congenital Generalized NIH-5 15/F None 115 0.8 2670 15 CongenitalGeneralized NIH-6 37/F None 25 0.6 1370 15 Congenital Generalized NIH-742/F Familial Gemfibrozil 40.3 3.6 1980 26 Partial UTSW-1 31/FFenofibrate 61.5 0.7 1702 8 Congenital Generalized UTS W-2⁵ 33/FGemfibrozil 12.3 2.4 1497 14 Acquired Generalized¹Fasting insulin, conversion factor to pmol/L: 7.15X (note that somepatients are on exogenous insulin therapy)²Conversion factor to nmol/mL: 0/08X³Residing metabolic rate⁴Obtained by measurements using dual-energy X-ray absorbtiometry wherethe measurements are 7-8% higher than underwater weighing technique.⁵Non-diabetic patient

All nine patients recruited into the study were females. Though thestudy was open to both genders, females tend to be recognized earlierand more frequently. Five of the nine patients had congenitalgeneralized lipodystrophy or the Seip-Beradinelli Syndrome. Thisdiagnosis was established with evidence of generalized fat loss sincebirth, in association with other clinical criteria (Online MendelianInheritance in Man, OMIM #269700; Garg et al., 1992). Three patientsappeared to have acquired generalized lipodystrophy with a history ofapparent fat loss in childhood. One of these patients (UTSW-2) developedgeneralized lipodystrophy with juvenile dermatomyositis. Another patient(NIH-7) had Dunnigan's familial partial lipodystrophy (OMIM #151660;Garg, 1999; and Cao et al., 2000).

Study Design

The study was designed as a prospective open-label study at the DiabetesBranch of National Institute of Diabetes, Digestive and Kidney Diseases(NIDDK), and at the University of Texas Southwestern (UT Southwestern)Medical Center at Dallas. Amgen Inc. (Thousand Oaks, Calif.) providedrecombinant methionyl human leptin (recombinant leptin) for the trial.Response of each patient was compared to her baseline state. Because ofthe rarity of lipoatrophy syndromes and the variability of the clinicalfeatures, it was not feasible to include a randomized placebo-treatedcontrol group. The institutional review boards of the NIDDK andUniversity of Texas Southwestern Medical Center approved the study.Informed written consent was obtained from the patient or the legalguardian.

Patients were evaluated as in-patients at the Clinical Center of theNational Institutes of Health and at the General Clinical ResearchCenter of the University of Texas Southwestern Medical Center beforetreatment and again after 1, 2 and 4 months of leptin therapy. Allpatients were on stable doses of concomitant medications for at least 6weeks before staring leptin. During the study, hypoglycemic drugs weretapered or discontinued as needed.

The goal in this study was to achieve near-physiological concentrationsof leptin in the plasma. The physiological replacement dose wasestimated to be 0.02 mg/kg/day for males of all ages, 0.03 mg/kg/day forfemales under 18 years and 0.04 mg/kg/day for adult females. Recombinantleptin was administered subcutaneously every 12 hours. It is importantto note that the replacement dose is approximately one tenth of the dosemost commonly used in obesity trials. Patients were treated with 50% ofthe replacement dose for the first month, 100% replacement dose the nextmonth and 200% replacement dose for the following two months. Theprimary end-points to determine efficacy of recombinant leptin weredetermined as Hemoglobin A_(1c) and fasting serum triglyceride levels.

Biochemical Analyses

Serum glucose and triglyceride levels were determined by standardmethods using automated Hitachi equipment (Boehringer Mannheim,Indianapolis, Ind.) and using Beckman Instrument (Beckman, Calif.).Hemoglobin A_(1c) was determined by ion-exchange high-pressure liquidchromatography (Bio-Rad Laboratories Inc., Hercules, Calif.). Serum freefatty acid (FFA) levels were determined with a commercial kit (Wako,Richmond, Va.). Serum insulin levels were determined by immunoassaysusing reagents provided by Abbott Imx Instrument (Abbott Park, Ill.) anda commercial kit (Linco Research, Inc., St. Charles, Mo.). Serum leptinlevels were determined by immunoassays using a commercial kit (LincoResearch, Inc. St. Charles, Mo.).

Procedures

Resting energy expenditure was measured using Deltatrac Equipment(Sensormedics, Yorba Linda, Calif.). The test was performed after anovernight fast for more than 8 hours in resting patients upon awakeningbetween 6 and 8 AM. Oral glucose tolerance test was performed after anovernight fast using 75 grams of dextrose. Serum glucose was measured at−10, 0, 30, 60, 90, 120 and 180 minutes of the glucose load.

A high-dose insulin tolerance test was performed using 0.2 IU/kg regularinsulin to assess insulin sensitivity. Insulin was administeredintravenously after an overnight fast. Samples for glucose werecollected at −10, 0, 5, 10, 15, 20 and 30 minutes of insulinadministration. K constant (the rate of glucose disappearance as areflection of total body insulin sensitivity) was calculated as the rateconstant for the fall in blood glucose after intravenous insulin usingfirst order kinetics (Harrision et al., 1976).

Body fat was determined using dual energy x-ray absorptiometer (DEXA,Hologic QDR 4500) (Hologic, Inc., Bedford, Mass.) (Lambrinoudaki et al.,1998). Axial TI weighted MR scans of the liver were obtained on a 1.5tesla scanner (General Electric Medical Systems, Milwaukee) (Abate etal., 1994). Liver volumes were calculated using the MEDx image analysissoftware package (Sensor Systems, Inc., Sterling, Va.), on a Sunworkstation. By placing a seed point for an edge following algorithm,tracings of the outer margins of the liver were made on individualcontiguous slices. The liver volumes were then computed based on thepixel area and slice thickness. Subjects participating at the NIH-sitewere asked to report their food intake in the last 3 days at baselineand at 4-months to calculate estimated daily food intake (Feskanich etal., 1993).

Statistical Analyses

Measurements are presented as mean±SEM. To compare study variablesduring various study periods, repeated measures analysis of variance wasused. Skewed data such as the triglyceride concentrations and thecalculated K constants were log-transformed. Paired t-test was employedto compare baseline data with various time points wherever applicable.Plasma glucose concentration during the oral glucose tolerance test werecompared using a 2-factor analysis of variance with study period andtime during the test modeled as repeated factors. Ninety-five percentconfidence intervals of the differences between the means were derivedfrom the analysis of variance and for the differences between the means(Hanh et al., 1991). Changes were considered statistically significantfor p<0.05. No adjustments for simultaneous comparisons were made forstatistical analyses of specific a priori hypotheses.

Results

Baseline Patient Characteristics

Eight of nine patients in the study were diabetic and all werehyperlipidemic (Table 1). All diabetic patients received pharmacotherapyprior to the study (Table 1 and 2) and 4 patients receivedpharmacotherapy for lipid management (Table 1). The average HbA_(1c) ofthe diabetic patients was 9.1±0.5% (normal: <5.6%). The meantriglyceride levels were elevated at 1405 mg/dL (range: 322-7,420 mg/dL;normal range: 35-155 mg/dL) [16 mmol/L, range: 3.6-8.7 mmol/L]. Freefatty acid (FFA) levels were increased about 3-fold from the upper limitof normal (1540±407 μmol/L; normal: 350-550 μmol/L). Six of the sevenNIH patients had fatty liver on ultrasound and enlarged livers onphysical exam. Three of the patients underwent liver biopsies and two ofthe three were diagnosed with non-alcoholic steatohepatitis based onhistopathological criteria (Manton et al., 2000; Berasain et al., 2000;Luyckx, et al., 2000).

The mean serum leptin concentration was 1.3±0.3 ng/mL at baseline(Table 1) which increased with therapy to 2.3±0.5 ng/mL at the end ofthe first month, 5.5±1.2 ng/ml at the end of the second month, and11.1±2.5 ng/mL at the end of the fourth month. Therefore, recombinantleptin administration at the doses used in this study resulted inapproximately normal serum leptin levels in these patients.

Effect of Leptin on the First Patient: A Case Example (FIG. 1)

The first patient treated in the study (NIH-1) is the most severelyaffected and her course is instructive in showing the dramatic effect ofleptin replacement in this population even after all other potentialtherapies have been extinguished. This patient was born healthy, butexperienced fat loss between age 10 and 12. She developed severehypertriglyceridemia at age 13 and diabetes at age 14. She was presentedto the NIH Clinical Center at age 15 with triglyceride levelsconsistently >10,000 mg/dL (>113 mmol/L) and diabetes with HbA_(1c) of9.5%. She had painful eruptive cutaneous xanthomata scattered throughoutthe body and massive hepatomegaly extending to the pelvic brim. Weeklyplasmapheresis therapy and Orlistat were added to alleviatehypertriglyceridemia (FIG. 1A) (Bolan et al.). Other remarkable clinicalfeatures included a voracious appetite (she reported eating in excess of3200 kcal/day) and a greatly elevated resting metabolic rate at 2010kcal/day, 180% of predicted. Over a four-month period, recombinantleptin caused a marked progressive improvement in hypertriglyceridemiaand hyperglycemia that allowed for discontinuation of plasmapheresis anddiabetes medications (FIG. 1A). The improvements in metabolic parameterswere accompanied by disappearance of cutaneous xanthomata. In addition,her liver volume decreased by 40% (from 4213 mL at baseline to 2644 mLat 4 months, shown in FIG. 1B).

Leptin Improved Metabolic Control in all Diabetic Lipoatrophic Patients

Prior to the initiation of leptin therapy, the eight diabeticlipoatrophic patients had poor metabolic control. With four months ofleptin replacement therapy, HbA_(1c) decreased by a mean of 1.9percentage points (95% CI, 1.1 to 2.7%, p=0.0012) (FIG. 2). Individualresponses of patients are shown in Table 3. It is notable that glycemiccontrol improved despite decreasing or discontinuing baselineanti-diabetes therapy (Table 2). TABLE 2 Changes in hypoglycemic therapyduring the study Hypoglycemic therapy during Hypoglycemic therapyPatient baseline period at 4-months of therapy NIH-1 Metformin (500 mgbid) None Acarbose (50 mg tid) NIH-2 Insulin (800 U/day) None NIH-3Insulin (40 U/day) None Metformin (500 mg tid) NIH-4 Insulin (1200U/day) None NIH-5 Insulin (3000 U/day) None NIH-6 Metaformin (500 mgtid) None NIH-7 Insulin (200 U/day) Insulin (60 U/day) Proglitazone (45mg qd) UTSW-1 Insulin (700 U/day) Insulin (300 U/day) UTSW-2 None NoneNondiabetic patient

The plasma glucose levels during the insulin tolerance test showedsignificant improvement at the end of 4 months compared to the baseline(FIG. 3A). The K-value (rate of glucose disappearance) increased from0.0071±0.0012 to 0.0169±0.0039 indicating improvement of whole-bodyinsulin sensitivity (p=0.035). Further, the oral glucose tolerance wasalso significantly improved compared to baseline (FIG. 3B).

At the end of four months of recombinant leptin therapy, the fastingtriglyceride levels fell by 60% (CI, 43 to 77%, p<0.001, FIG. 4). Duringthis same period, fasting free fatty acids fell from 1540±407 μmol/L to790±164 μmol/L (p=0.045). Individual responses are shown in Table 3.TABLE 3 Patients' metabolic parameters during different stages oftherapy HbA_(IC) Triglycerides¹ Free fatty acids² (%) (mg/dL) (pmol/L)Month³ Patients 0 1 2 4 0 1 2 4 0 1 2 4 NIH-1 8.6 7.6 7.4 7.0 7420 64401632 1214 3977 3517 2216 1701 NIH-2⁴ 9.8 8.3 7.4 10.0 633 523 471 4052922 1452 1372 1244 NIH-3 9.3 7.8 8.4 7.9 450 579 233 281 919 368 451454 NIH-4 7.6 6.7 6.1 5.0 322 232 160 106 1838 1388 866 446 NIH-5 9.59.4 6.5 6.1 913 427 143 123 1066 1842 723 629 NIH-6 9.2 8.6 7.2 7.4 663355 242 303 1672 1367 1315 428 NIH-7 9.5 8.4 7.4 6.6 802 366 295 215 384315 306 345 UTSW-1 9.5 8.1 7.5 7.3 995 827 383 192 560 360 525 560UTSW-2⁵ 5.4 4.8 5.0 5.1 447 656 276 424 520 630 1690 1310¹Fasting plasma triglyceride levels, conversion factor to mmol/L:0.1129X, normal 35-155 mg/dL²Fasting free fatty acid levels, normal 135-550 pmol/L³Month of therapy, 0 refers to baseline evaluation period⁴This patient had noncompliance between 3^(rd) and 4^(th) months oftherapy. After two months of strict compliance as documented by vials ofmedication used, the reported parameters were respectively: 7.3%, 283mg/dL and 799 pnol/L⁵Non-diabetic patientChanges in Liver Volume, and Liver Function Tests

Baseline mean liver volume was 3097±391 mL (about 4-fold elevatedcompared to age and sex-matched normal weight individuals). Leptindecreased the liver volume by an average of 28% (CI, 20 to 36%) frombaseline. The mean decrease in liver volume was 987 mL (CI, 546 to 1428mL, p=0.0024). The improvement in liver size was associated withimprovement in liver function tests. Baseline alanine-transaminaseconcentrations decreased from 66±16 U/L to 24±4 U/L at the end of 4months (p=0.023). Likewise, serum aspartic-transaminase concentrationswere 53±12 U/L at baseline and 21±2 U/L at the end of 4-months (p=0.03).

Changes in Energy Balance

Self-reported daily caloric intake was greatly reduced from a baselineof 2680±250 kcal/day to 1600±150 kcal/day (p=0.005, n=7). There was aparallel decrease in the measured resting metabolic rate 1920±150kcal/day to 1580±80 kcal/day (p=0.003, n=9).

All but one (NIH-3) subject had weight loss at the end of 4 months. Themean weight loss was 3.6±0.9 kg with a range between −1.7 and 7.3 kg. Animportant fraction of weight loss (50-65%) can be attributed to loss ofliver weight.

Tolerability and Adverse Events

No skin reactions at injection sites were reported or observed. Therewere no trends towards adverse effects on routine biochemical orhematological parameters. Patients NIH-1 had a severe episode of nauseaand vomiting after the first dose. Patient NIH-6 had exacerbation ofhypertension after the second dose associated with flushing.

Patient NIH-7 was hospitalized due to streptococcus infection during thethird month of therapy. None of these events recurred with continuedtherapy.

Discussion

In this study, leptin replacement led to clear and dramatic metabolicbenefits in a group of patients with lipodystrophy and leptindeficiency. During the study, replacement with recombinant leptinresulted in 1.9 percentage point improvement in HbA_(1c), which ispredicted to decrease the relative risks to develop retinopathy by ˜22%in the diabetic population (UK PDS, 1998). Furthermore, triglyceridelevels fell by 60%, which is predicted to decrease the relative risk forcardiovascular events in the general population by 35-45% (Kreisberg,1998; Garg, 2000)

These results provide a novel insight into the mechanisms of action ofleptin. Leptin signal appears to regulate total body insulin sensitivityand triglyceride levels in addition to its known role in the control ofenergy homeostasis. This study is the first evidence that leptinfunctions as an insulin-sensitizing and insulin-sparing agent in vivo inhumans.

Although a randomized study design was not employed, the weight ofevidence suggests that the improved metabolic control was caused byleptin rather than improved compliance associated with participation ina study. First, the magnitude and reproducibility of the improvement ofHbA_(1c) are most consistent with a drug effect rather than a placeboeffect. Despite the heterogeneity of the patients included in our study,we observed a uniform improvement in metabolic control in all thediabetic patients. There was evidence of noncompliance in patient NIH-2,explaining the worsening of her HbA_(1c) between 2 and 4 months that wascorrected with prolonged therapy (Table 3). This patient-improved drugwithdrawal is strong evidence that the effect on improved HbA_(1c)levels is due to leptin administration.

Effect of Leptin on Food Intake

It is recognized that limiting caloric intake in lipoatrophic diabetesimproves glucose and lipid abnormalities (Trygstad et al., 1977).However, patients have difficulty complying with meal limitations due totheir appetite. Leptin clearly reduced food intake in these patients. Alimited study was carried out with Patient NIH-1 to determine thecontribution of deceased food intake on the metabolic parameters. In thehospital, she underwent 9 days of leptin withdrawal with caloric intakeclamped at pre-withdrawal levels. Despite being on a steady diet, herfasting insulin, triglyceride and glucose concentrations increasedwithin 48 hours. These observations indicate that leptin has effects oninsulin sensitivity and triglyceride metabolism independent of itseffects on food intake. Similar data using pair-feeding experiments inlipoatrophic mice with or without leptin administration have beenreported (Shimomura et al., 1999; Ebihara et al., 2001).

Correlation with Mouse Models

The various mouse models of lipoatrophy suggested that the absence ofadipose tissue is the cause of insulin resistance in this syndrome(Burant et al., 1997; Moitra et al., 1998; Shimomura et al., 2000). Thedemonstration that transplantation of adipose tissue into lipoatrophicmice dramatically ameliorates insulin resistance and improves metaboliccontrol provides strong support for this hypothesis (Gavrilova et al.,2000). However, it remained unclear why adipose tissue was required tomaintain whole body insulin sensitivity. The observations and theresults discussed above, together with Shimomura et al supra, suggestthat the majority of the regulatory action of adipose tissue on wholebody insulin sensitivity act through leptin.

Possible mechanism of how leptin regulates both insulin sensitivity andlipid metabolism may be based on SREBP1c, a transcription factorstimulating lipogenesis. In the liver, SREBP1c is upregulated byhyperinsulinemia seen in lipoatrophy. Leptin deficiency andhyperinsulinemia cause down-regulation of insulin-receptor substrate,IRS-2, impairing insulin action and increasing hepatic glucose output.The increased lipogenesis and hepatic glucose output create a viciouscycle. Increased tissue lipid levels are associated with decreased wholebody insulin sensitivity and thus more hepatic glucose output.Replacement of leptin is shown to correct this vicious cycle. While therate of triglyceride synthesis was not studied in humans withlipoatrophy, the indirect calorimetric studies provide some evidencethat lipogenesis may in fact be dysregulated (Arioglu et al., 2000).Another observation was the decline in resting energy expenditure in thepatients treated in this study. This may be due to decreased food intakeresulting in reduced diet-induced thermogenesis.

Leptin: An Anti-Steatosis Hormone

It has been reported that leptin administration in Zucker rats leads tocorrection of steatosis in a variety of organs that act as lipidaccumulation sites; such as the islet cells of the liver or heart cells(Unger, 1995; Unger et al., 1999). The lipid accumulation outside of theadipocytes may be a spill over phenomenon resulting from the adipocyteshaving reached maximum capacity to store triglycerides. Inlipodystrophy, these organs are the only sites that can store lipids.Leptin treatment in mice with lipodystrophy causes a dramatic fall inhepatic triglyceride stores. In parallel, leptin therapy in humans withlipodystrophy causes a remarkable, highly significant reduction in livervolumes.

Timing for Leptin Replacement

The concept that adipose tissue is an endocrine organ was stronglysupported by the discovery of leptin. Leptin has effects, both directand/or indirect, on the key organs of metabolism, including the brain,liver, muscle, fat and pancreas. Leptin certainly is not the onlycirculating adipocyte signal. For example, another adipocyte hormone isadipocyte specific complement related protein (ACRP)30/Adiponectin/AdipoQ which seems to be important in inducing fatoxidation in the muscle and liver (Yamauchi et al., 2001; Fruebis etal., 2001; Berg et al., 2001). Lack of adipocytes should result indeficiency of all fat-derived signals known and yet to be discovered,thus contributing to many of the abnormalities seen in syndromescharacterized by absence of fat. This study is the first human studylooking at the metabolic efficacy of replacing a fat-derived hormone ina state of fat deficiency. It appears that leptin deficiency is themajor contributor (but probably not the only one) to the metabolicabnormalities seen in association with lipoatrophy. As such, this studyunderscores an important reason to consider leptin replacement therapyin humans; namely severe lipodystrophy.

Example III

The amino acid sequence for mature, recombinant methionyl human leptinis presented herein as SEQ ID NO. 1, where the first amino acid of themature protein is valine (at position 1) and a methionyl residue islocated at position-I (herein called rHu-Leptin 1-146, SEQ ID No. 1).  V P I Q K V Q D D T K T L I K T I V T R I N D I S H T Q S V S S K Q KV T G L D F I P G L H P I L T L S K M D Q T L A V Y Q Q I L T S M P S RN V I Q I S N D L E N L R D L L H V L A F S K S C H L P W A S G L E T LD S L G G V L E A S G Y S T E V V A L S R L Q G S L Q D M L W Q L D L SP G C

Alternatively, one may use a natural variant of human leptin, which has145 amino acids, and, as compared to rHu-Leptin 1-146, has a glutamineabsent at position 28, presented below (herein called rHu-Leptin 1-145,SEQ ID NO. 2, wherein the blank (“*”) indicates no amino acid).   V P IQ K V Q D D T K T L I K T I V T R I N D I S H T * S V S S K Q K V T G LD F I P G L H P I L T L S K M D Q T L A V Y Q Q I L T S M P S R N V I QI S N D L E N L R D L L H V L A F S K S C H L P W A S G L E T L D S L GG V L E A S G Y S T E V V A L S R L Q G S L Q D M L W Q L D L S P G C

Other examples of leptin proteins, analogs, derivatives, preparations,formulations, pharmaceutical composition, doses, and administrationroutes have previously been described in the following PCT Applicationsand are hereby incorporated by reference as if fully set forth herein.PCT International Publication Number WO 96/05309; WO 96/40912; WO97/06816, WO 00/20872; WO 97/18833; WO 97/38014; WO 98/08512 and WO98/28427.

Leptin proteins, analogs and related molecules are also reported in thefollowing publications; however, no representation is made with regardto the activity of any composition reported.

U.S. Pat. Nos. 5,521,283; 5,525,705; 5,532,336; 5,552,522; 5,552,523;5,552,524; 5,554,727; 5,559,208; 5,563,243; 5,563,244; 5,563,245;5,567,678; 5,567,803; 5,569,743; 5,569,744; 5,574,133; 5,580,954;5,594,101; 5,594,104; 5,605,886; 5,614,379; 5,691,309; 5,719,266 (EliLilly and Company);

PCT WO96/23513; WO96/23514; WO96/23515; WO96/23516; WO96/23517;WO96/23518; WO96/23519; WO96/34111; WO 96 37517; WO96/27385; WP97/00886; EP 725078; EP 725079; EP 744408; EP 745610, EP 835879 (EliLilly and Company);

PCT WO96/22308 (Zymogenetics);

PCT WO96/31526 (Amylin Pharmaceuticals, Inc.)

PCT WO96/34885; WO 97/46585 (SmithKline Beecham, PLC);

PCT WO 96/35787 (Chiron Corporation);

PCT WO97/16550 (Bristol-Myers Squibb);

PCT WO 97/20933 (Schering Corporation)

EP 736599 (Takeda);

EP 741187 (F. Hoffman La Roche).

To the extent these references provide for useful leptin proteins oranalogs, or associated compositions or methods, such compositions and/ormethods may be used in conjunction with the present methods. With theabove provisos, these publications are herein incorporated by reference.

Example IV

A standard enzyme-linked immunosorbent assay (ELISA) may be used todetermine leptin levels in the serum of lipoatrophic patients accordingto one embodiment of the present invention. The ELISA method may use apurified rat monoclonal anti-rmetHu-Leptin antibody for capturing leptinfrom serum. Affinity purified rabbit anti-rmetHu-leptin polyclonalantibody conjugated to horseradish peroxidase may also be used to detectcaptured leptin. The limit of detection of the said assay using theseantibodies may be in the range of 0.5-0.8 ng/ml. Although certainantibodies may have been used, preferred antibodies are those whichspecifically react with native human leptin, and are sensitive to detectleptin quantities of equal to or below 5 ng/ml serum.

Preferably, the timing for determining the baseline leptin levels in apatient is after an 8-12 hour fast such as during morning hours.Baseline leptin levels may not be confounded by raising levels, such asafter a meal, or due to sleep cycle rise in leptin seen in mostindividuals (e.g., 3:00 a.m. rise in leptin levels). Such baselinelevels may be used, such as observation of nocturnal elevation of leptinlevels, but those levels should be compared at similar levels insimilarly situated patients.

Based on the above data, a method of determining predisposition oflipoatrophic patients to treatment with leptin can be performed bydetermining the leptin level corresponding to the serum leptinconcentration and ascertaining that the serum leptin concentration isabout 4 ng/ml or less.

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1. A method of treating a human patient with a condition of lipoatrophy,which comprises administering to the patient an effective dose ofleptin, leptin analog or leptin derivative.
 2. The method of claim 1,wherein said leptin, leptin analog or leptin derivative is administeredtogether with a pharmaceutically acceptable carrier.
 3. The method ofclaim 1, wherein said leptin, leptin analog or leptin derivative isadministered in a pharmaceutically acceptable diluent.
 4. The method ofclaim 1, wherein the patient has a leptin level of 4 ng/ml or lessbefore administration of leptin, leptin analog, or leptin derivative. 5.The method of claim 4, wherein the patient has a leptin level of 2 ng/mlor less before administration of leptin, leptin analog, or leptinderivative.
 6. The method of claim 1, wherein the patient has anacquired form of lipoatrophy.
 7. The method of claim 6, wherein thepatient is HIV positive.
 8. The method of claim 7, wherein the acquiredform of lipoatrophy is related to treating the HIV positive patient withhighly active antiretroviral therapy (HAART).
 9. The method of claim 1,wherein the patient has a genetic form of lipoatrophy.
 10. The method ofclaim 9, wherein the genetic form of lipoatrophy is congenitalgeneralized lipoatrophy.
 11. The method of claim 1, wherein thecondition of lipoatrophy comprises metabolic abnormalities.
 12. Themethod of claim 11, wherein the metabolic abnormalities are selectedfrom a group consisting of hyperglycemia, dyslipidemia, hyperlipidemia,hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascularrestenosis, and insulin resistance.
 13. The method of claim 11, whereinthe metabolic abnormalities comprise diabetes.
 14. The method of claim11, wherein the metabolic abnormalities comprise insulin resistance. 15.The method of claim 11, wherein the metabolic abnormalities comprisehypertriglyceridemia.
 16. The method of claim 1, wherein the conditionof lipoatrophy comprises hepatomegaly.
 17. The method of claim 1,wherein the condition of lipoatrophy comprises an abnormality in thedistribution of fat tissue.
 18. The method of claim 1, wherein saidleptin, leptin analog, or leptin derivative is administeredsubcutaneously.
 19. The method of claim 18, wherein the leptin isselected from the group consisting of recombinant human leptin of SEQ IDNO: 1 and SEQ ID NO:
 2. 20. The method of claim 19, wherein said leptinis administered together with a pharmaceutically acceptable carrier. 21.The method of claim 19, wherein said leptin is administered in apharmaceutically acceptable diluent.
 22. (canceled)
 23. The method ofclaim 1, wherein said leptin is recombinant human leptin.
 24. The methodof claim 23, wherein said recombinant human leptin is SEQ ID NO:
 1. 25.A method of determining a predisposition of a lipoatrophic patient torespond to treatment with leptin, leptin analog, or leptin derivative,the method comprising: (a) determining a leptin level in the patientprior to said treatment; and (b) ascertaining whether the leptin levelis less than or equal to approximately 4 ng/ml.
 26. The method of claim25, wherein said patient is a male and said leptin level is less than orequal to approximately 2 ng/ml prior to treatment.
 27. The method ofclaim 25, wherein said patient is a female.
 28. A method of determininga predisposition of a lipoatrophic patient to respond to treatment withleptin, leptin analog, or leptin derivative, the method comprising: (a)determining a leptin level in the patient prior to said treatment; and(b) ascertaining whether the leptin level of (i) a male patient is lessthan or equal to approximately 2 ng/ml, or (ii) a female patient is lessthan or equal to approximately 4 ng/ml.
 29. A method for treatinglipoatrophy, comprising a pharmaceutical regimen comprising acombination of protease inhibitor and leptin, leptin analog, or leptinderivative.
 30. A method for treating lipoatrophy, comprising apharmaceutical regimen comprising a combination of leptin, leptinanalog, or leptin derivative and at least one compound selected from thegroup consisting of thiazolidinediones, fibrates, statins and metformin.31. A method of treating a human with metabolic abnormalities associatedwith lipoatrophy, comprising administering leptin, leptin analog, orleptin derivative.
 32. An improved kit for determining thepredisposition of a human patient with lipoatrophy to respond totreatment with leptin, leptin analog or leptin derivative, theimprovement comprising means for determining whether the leptin level ofthe patient prior to said leptin treatment is: (i) less than or equal toapproximately 2 ng/ml if said patient is male, or (ii) less than orequal to approximately 4 ng/ml if said patient is female.
 33. A methodof treating a human patient with a condition of lipoatrophy, whichcomprises administering to the patient an effective dose of a leptin, aleptin analog or a leptin derivative, wherein said leptin, leptinanalog, or leptin derivative is delivered to the patient using a vectorcomprising nucleic acid sequences encoding said leptin, leptin analog,or leptin derivative, respectively.